KIF13B-mediated VEGFR2 trafficking is essential for vascular leakage and metastasis in vivo

The cancer cells secrete VEGF, which induces vascular leakage and metastasis. Inhibition of VEGFR2 trafficking to the cell surface prevents receiving VEGF, vascular leakage, and cancer metastasis.

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1) I feel that each section of the introduction is not well connected logically. In addition, although the description of each data is proper, the connection between the data is weak. Furthermore, the description in the "introduction" and "discussion" sections are often redundant. On the other hand, necessary information in the discussion is missing. For instance, the authors described in the introduction "Thus, it can be a safer alternative strategy to target VEGF/VEGFR2 pathway for angiogenesis-related diseases such as cancer. " Safer than what? I guess the authors want to claim the potential advantage of KAI peptide for patient treatment than current clinical interventions. If so, the authors should mention VEGF inhibitors which is widely used in the clinics. The authors especially mentioned the importance of VEGFR2 trafficking on AKT and Src signaling. Does the treatment of ECs with KAI suppress those signaling pathways?
2) The authors showed the role of KIF13B on VEGF-induced endothelial sprouting, VEGF-induced vascular leakage, and tumor metastasis, which highlight different endothelial function. Design and result interpretation are fine, however, they do not connect nicely in the single manuscript from the logical point of view. How about discuss the connection of each data in the discussion section? 3) Abbreviations should be defined in the manuscript. For instance, KAI, IHC, and so on. Additionally, the gene name should be described according to the mouse gene nomenclature. For instance, "KIF13B" should be "Kif13b" 4) The authors previously showed that KIF13B binds to VEGFR2 and controls VEGFR2 cell surface presentation. Then, they developed a peptide inhibitor which blocks binding of KIF13B with KIF13B. What is the evidence that treatment of ECs with KAI suppresses VEGFR2 cell surface presentation? 5) In the method section, the authors must describe each information more precisely. What is the catalog number of each antibody? The name of animal ethics committees should be described. What is the dilution of Matrigel? What is the company provided VEGF? Mouse VEGF? Human VEGF? VEGF-A? VEGF-C? VEGF165? In the method section, VEGF concentration for matreigel plug assay was 40ng/ml. On the other hand, it is described as 50 ng/ml in the figure legend. What kind of statistical analysis the authors applied for each experiment? 6) Given the phenotype of EC specific Kif13b KO mice and KAI treated mice in the tumor metastasis assay, specificity of KAI peptide should be clarified. If KAI peptide is treated with EC specific Kif13b KO mice, what would be happened?
Reviewer #2 (Comments to the Authors (Required)): Here, Waters et al. study the role for the kinesin-like motor protein KIF13B, which they previously have shown regulates intracellular transport of VEGFR2 to the cell surface. Using a newly generated endothelial-specific KIF13B deletion mouse model, the authors now show that in the absence of endothelial KIF13b, sprouting angiogenesis, lung colonization of B16F10 tumor cells injected in the tail vein, and vascular leakage are all suppressed. There is also delayed growth and vascularization of subcutaneous B16F10 tumors. The results phenocopy those seen with a KIF13B-derived peptide described by Dr. Yamada in several publications, which blocks the interaction between KIF13B and VEGFR2. This is a well conducted and presented study confirming an important role for VEGFR2 in control of the vascular barrier. It moreover highlights the potential of the KIF13Bbased peptide in development of novel therapeutics to regulate pathological angiogenesis. I have no criticisms.

st Authors' Response to Reviewers
September 16, 2021 Reviewer #1: 1) I feel that each section of the introduction is not well connected logically. In addition, although the description of each data is proper, the connection between the data is weak. Furthermore, the description in the "introduction" and "discussion" sections are often redundant. On the other hand, necessary information in the discussion is missing. For instance, the authors described in the introduction "Thus, it can be a safer alternative strategy to target VEGF/VEGFR2 pathway for angiogenesis-related diseases such as cancer. "Safer than what? I guess the authors want to claim the potential advantage of KAI peptide for patient treatment than current clinical interventions. If so, the authors should mention VEGF inhibitors which is widely used in the clinics. The authors especially mentioned the importance of VEGFR2 trafficking on AKT and Src signaling. Does the treatment of ECs with KAI suppress those signaling pathways?
Thank you for your helpful comments. We edited the text mainly between each paragraph to connect each paragraph logically. For example, between the 1 st paragraph and 2 nd paragraph of the introduction, we added, "Besides the total amount of VEGFR2 described above, the availability of VEGFR2 on the cell surface is a critical factor to regulate VEGFR2 signaling, as VEGFR2 receives VEGF on the cell surface. On the endothelial cell surface,…" As we thoroughly edited the text, we left track change in the manuscript. Also, to describe what KAI is safer than, we wrote, "Concerning the systemic effect of anti-VEGF therapy (Ferrara and Adamis, 2016;Crawford and Ferrara, 2009), selectively targeting VEGFR2 trafficking can be a safer alternative strategy to target VEGF/VEGFR2 pathway for angiogenesis-related diseases such as cancer." Regarding the signaling molecules under VEGF/VEGFR2, we revised them as "activation of downstream signaling such as SRC and ERK (Lanahan et al., 2010;Simons et al., 2016). "We have tested the effect of KAI on downstream signaling molecules such as SRC. However, we would like to keep the data for the next manuscript, as it is one of the most important data for the next one.

2) The authors showed the role of KIF13B on VEGF -induced endothelial sprouting, VEGF-induced vascular leakage, and tumor metastasis, which highlight different endothelial function. Design and result interpretation are fine, however, they do not connect nicely in the single manuscript from the logical point of view. How about discuss the connection of each data in the discussion section?
In the discussion section, we discussed the connection between endothelial permeability and angiogenesis. Please see the 4 th paragraph and 5 th paragraph in the discussion section.
3) Abbreviations should be defined in the manuscript. For instance, KAI, IHC, and so on. Additionally, the gene name should be described according to the mouse gene nomenclature. For instance, "KIF13B" should be "Kif13b" In the revised manuscript, we carefully spell out the abbreviations when they first appear. Also, we followed the rule of the mouse gene nomenclature. We added the catalog number of the antibodies in the methods section. Note, for the data in this manuscript, we used the rabbit polyclonal antibody against KIF13B from Sigma (HPA025-023), which was discontinued. The new antibody from Sigma with a different lot number does not work well. We are currently using homemade mouse monoclonal antibody for recent data. We added the name of the animal ethics committee in the methods section, "Animal Care Committee administered through the Office of Animal Care and Institutional Biosafety at UIC." Human recombinant VEGF-A165 is from Peprotech, we used 40 ng/ml VEGF in Matrigel plug assay. We corrected the figure legend. Matrigel was used without dilution. We used Graph Pad Prism 9 (GraphPad Software) for statistical analysis. We analyzed data by one-way ANOVA followed by post hoc Bonferroni multiple comparisons. To compare two samples, we used Student's t-test. We added one paragraph of Statistical analysis in the methods section.

6) Given the phenotype of EC specific Kif13b KO mice and KAI treated mice in the tumor metastasis assay, specificity of KAI peptide should be clarified. If KAI peptide is treated with EC specific Kif13b KO mice, what would be happened?
Thank you for the great advice. We tested the effect of KAI treatment in lung metastasis in Kif13b iECKO and Kif13b WT . We added the data in Fig. 5. "Consistent with Fig. 1, KAI treatment significantly reduced the number of metastasis in Kif13b WT compared with treatment with ctrl peptide (Fig. 5A, B). Consistent with Fig. 4 (Fig.  5A, B). This data suggests that the target of KAI is mainly KIF13B and VEGFR2 in EC." Then, we discussed in the discussion section, "Metastasis is multiple- (Fig. 5) Thank you for submitting your revised manuscript entitled "KIF13B Mediated VEGFR2 Trafficking Regulates Vascular Leakage and Metastasis in vivo". We would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines, as well as addressing the remaining points made by Reviewer #1.

step events, such as escape of cancer cells from the primary tumor, intravasation of cancer cells into the bloodstream, survival of cancer cells in the bloodstream, extravasation of cancer cells through vascular barrier at the distal site, and colonization of cancer cells at the distal site. This experimental setting skips initial steps and starts from the injection of the cancer cells into the bloodstream. And the inhibitory effect of KAI was observed only in the presence of KIF13B in EC
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Thank you for this interesting contribution, we look forward to publishing your paper in Life Science Alliance. According to previous my comments, the authors address the raised issues. Importantly, data in Figure 5 strengthen the authors' conclusion. Date is fine. The discussion part was improved very much. However, I still feel the introduction section is not connected well and should be improved. I suggest to ask a specialist in the field to edit the introduction part. The information regarding statistical analysis is missing. What kind of statistical analysis was applied for each experiment? This is the serious problem. Those points must be fixed before publication.
3) The authors defined vascular endothelial growth factor A as "VEGF-A". However, in the most of the part of the manuscript, the authors' described as VEGF.